Cloning and characterization of two α-glucosidases from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis> DSM20083

Two alpha-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both alpha-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-alpha-glucoside. The K(m) for pnp-alpha-glucoside was 1.05 and 0.47 mM and the V(max) was 228 and 113 U mg(-1) for AglA and AglB, respectively. Using pnp-alpha-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37 degrees C. For AglB, values of pH 6.8 and 47 degrees C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant alpha-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.     

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.     

Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey

Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.     

Localization and Characterization of alpha-Glucosidase Activity in Brettanomyces lambicus

Brettanomyces lambicus was isolated and identified from a typical overattenuating Belgian lambic beer and exhibited extracellular and intracellular alpha-glucosidase activities. Production of the intracellular enzyme was higher than production of the extracellular enzyme, and localization studies showed that the intracellular alpha-glucosidase is mostly soluble and partially cell wall bound. Both intracellular and extracellular enzymes were purified by ammonium sulfate precipitation, gel filtration (Sephadex G-150, Sephadex G-200, Ultrogel AcA-44), and ion-exchange chromatography (sulfopropyl-Sephadex C-50, (carboxymethyl-Sephadex C-50). The intracellular alpha-glucosidase exhibited optimum activity at 39 degrees C and pH 6.2. The extracellular enzyme exhibited optimum catalytic activity at 40 degrees C and pH 6.0. The molecular masses of purified intracellular and extracellular alpha-glucosidases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 72,500 and 77,250, respectively. For both enzymes there was a decrease in the rate of hydrolysis with an increase in the degree of polymerization, and both enzymes hydrolyzed dextrins isolated from lambic wort (degrees of polymerization, 3 to 9 and more than 9). The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose for the intracellular enzyme were 0.9, 3.4, and 3.7 mM, respectively. The K(i) values for both enzymes were between 28.5 and 57 muM for acarbose and between 7.45 and 15.7 mM for Tris. These enzymes are probably involved in the overattenuation of spontaneously fermented lambic beer.     

Soluble and membrane-bound neutral alpha-glucosidase from the human kidney

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.     

Neutral alpha-glucosidase in granule fractions from guinea pig polymorphonuclear leukocytes: enzymic characterization and comparative studies with monoclonal antibodies

Neutral alpha-glucosidase was partially purified from granular fractions isolated from guinea pig polymorphonuclear leukocytes (PMNL). The native enzyme had a high molecular weight, about 417,000, with a subunit of 43,000. The purified enzyme hydrolysed 4-methylumbelliferyl alpha-glucoside and maltose, but not isomaltose, trehalose, and glycogen. The enzyme was strongly inhibited by bromoconduritol and castanospermine, but only slightly by turanose. Monoclonal antibodies which can bind specifically to the enzyme were prepared by immunizing mice with the partially purified enzyme. Hybridomas producing the monoclonal antibodies were selected by an enzyme-linked immunosorbent assay. The seven monoclonal antibodies were found to react with the enzyme from PMNL, but not with the glycoprotein-processing alpha-glucosidase isolated from liver microsomes nor with the macrophage enzyme. The results indicated that PMNL contain a particulate neutral alpha-glucosidase enzymologically and immunologically distinct from other alpha-glucosidases.     

Novel alpha-glucosidase from Aspergillus nidulans with strong transglycosylation activity

Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.     

Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.     

Purification and characterization of a novel fungal alpha-glucosidase from Mortierella alliacea with high starch-hydrolytic activity

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable alpha-amylase activity. Here, a alpha-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal alpha-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55 degrees C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-alpha-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50 degrees C.     

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.     

Change in maltose- and soluble starch-hydrolyzing activities of chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae

The chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae, which has high activity toward not only maltooligosaccharides but also soluble starch and has high activity toward maltooligosaccharides but faint activity toward soluble starch, respectively, were constructed by shuffling the C-terminal regions where low homology is observed between the two enzymes. The chimera genes were expressed in Pichia pastoris to produce and secrete the enzymes that have predicted molecular masses in the culture medium. The two chimeric M. javanicus alpha-glucosidases, of which the N- and C-terminal regions are substituted for those of A. oryzae, respectively, decreased in soluble starch-hydrolyzing activity, however, increased in maltose-hydrolyzing activity by 2.1 and 4.9 times higher than that of the native form of M. javanicus alpha-glucosidase, respectively. The chimeric enzymes changed on the V(max) values for maltose significantly, whereas the K(m) values were similar to that of the native enzyme.     

Molecular and physiological role of the trehalose-hydrolyzing alpha-glucosidase from Thermus thermophilus HB27

Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative alpha-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was alpha,alpha-1,1-trehalose, a new feature among alpha-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (alpha-1,2 linkage), nigerose and turanose (alpha-1,3), leucrose (alpha-1,5), isomaltose and palatinose (alpha-1,6), and maltose (alpha-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous alpha-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the alpha-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the alpha-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the alpha-glucosidase), indicating that the alpha-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.     

Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III

Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.     

Purification and characterization of a new type of alpha-glucosidase from Paecilomyces lilacinus that has transglucosylation activity to produce alpha-1,3- and alpha-1,2-linked oligosaccharides

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.     

Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K(m)) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.     

A novel alpha-glucosidase from the moss Scopelophila cataractae

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Similar distribution of the activities of neutral alpha-glucosidase (gamma-amylase) and glucose-6-phosphatase in subcellular fractions from rat and trout livers]

alpha-glucosidases are cellular enzymes, able to split the polysaccharides into glucose. In subcellular fractions from rat and trout hepatocytes, the distribution patterns of neutral alpha-glucosidase and of glucose-6-phosphatase appear to be very similar, i.e., closely linked to the endoplasmic reticulum, and are somewhat related to the particular glycogen. The data suggest a probable role of neutral alpha-glucosidase in cell physiology and in carbohydrates metabolism.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.